In recent decades our understanding of the gut microbiota has evolved from one of a host-pathogen relationship resulting in disease (eg. gastroenteritis, H pylori ulcers), to one comprising a much more complex and nuanced system of host and commensals working together to execute the multiple functions of the gut. These functions include not only the absorption of nutrients, but also protection from gut pathogens, regulation of the immune system to prevent inflammatory and allergic conditions, and involvement in the endocrine system. In this review we present the current state of knowledge on the makeup and role of the gut microbiota, specifically in infant health and disease, and consider the potential for promoting health through manipulation of the microbiota.

What does the normal gut microbiota consist of?

While the gut microbiome includes fungi, viruses and protozoans in addition to prokaryotes, research to date has focused primarily on the makeup and role of the bacterial microbiome.¹ Identification of the microbiome was previously limited by the sensitivity of culture techniques. However, recent technological advances, including DNA sequencing and metagenomics, have allowed the identification of many more previously undetected species and have increased the known number of species in the gut microbiota approximately 100-fold, to a currently estimated 15-36,000 different species.²

16S ribosomal RNA genes are unique to bacteria, and targeted sequencing of these short DNA segments allows identification of the different bacterial species without requiring them to be cultured. Metagenomics refers to whole DNA sequencing from a sample (eg. faecal, or tissue biopsy) with subsequent analysis allowing not only species identification, but also identification of gene function from the whole bacterial population (eg. antibiotic resistance, enzyme expression). 

The most common bacteria found in the healthy adult human gut are members of the phyla Firmicutes (eg. Lactobacillus, Streptococcus, Clostridiae, Ruminococcus, Enterococcus, Staphylococcus) and Bacteroidetes (with species including Bacteroides fragilis). The phyla Actinobacteria (including multiple Bifidobacterium species) and Verrucomicrobia are also prominent, as well as lower concentrations of other organisms, including known pathogenic bacteria such as Campylobacter jejuni, Salmonella enterica, Vibrio cholera and Escherichia coli.³

We know that bacteria vary both in concentration and content, depending on location within the gut (oesophagus, stomach, intestines; mucosal versus luminal). Most studies examining intestinal microbiota use faecal analysis which reflects luminal bacteria; however in some situations the mucosal bacteria may be more relevant. Study is limited by the need to obtain biopsy specimens).

Additionally, the ‘normal’ microbiota of the intestine varies significantly between individuals of the same community and within individuals over time, constantly responding to changes and maintaining homeostasis. The greatest interindividual variability occurs in infancy.

The intestinal microbiota in infants

The gut of the foetus is thought to be sterile and bacterial colonisation starts from the moment of birth. Total bacterial numbers steadily increase and reach levels of 109 CFUs/g of faeces by one week of age.⁴ Early colonisers are facultative anaerobes which can thrive in the relatively oxygen rich environment of the newborn gut. These consume oxygen thereby modifying the intestinal environment and allow colonisation by obligate anaerobes by the first week of life. The acquired flora is very diverse, including Staphylococci (S aureus, S epidermidis), Streptococci (S faecalis, non-haemolytic strep), Enterobacteriaciae (E coli, Klebsiella aerogenes, Proteus mirabilis and Enterobacter cloacae) and a variety of Bifidobacteria species (B bifidum, B infantis, B breve, B longum).⁴ Bifidobacteria and Enterobacteriacieae species are dominant in infants in most studies. 

There are many factors that affect the pattern of colonisation of the infant gut and the development of the term infant gut microbiome from birth is an area of considerable interest. We know that the mode of delivery (vaginal versus caesarean section) plays an important role in the early acquisition of gut bacteria; a vaginally delivered infant tends to acquire its microbiota from its mother’s vaginal and faecal flora; an infant delivered by caesarean section acquires its microbiota from environmental bacteria, eg. skin commensals. Several studies have shown that mode of delivery has an independent effect on the makeup of the infantile intestinal microbiota, with differences established in the first days of life and persisting for six months or even several years of age. These differences include a delay in anaerobic colonisation, a lower number of Bifidobacteria species and higher concentrations of C difficile, Staphylococcus species and other Clostridia and Bacteroides species in infants born by caesarean section compared to infants born by vaginal delivery.^5,6,7,8,9

The mode of infant feeding also has an influence on the establishment of the intestinal microbiome. Human milk contains its own probiotic microbiota which is transmissible to the infant gut (primarily Proteobacteria and Firmicutes), antimicrobials (antigen specific IgAs and non-specific bacteriocides including lactoferrin and lysozyme) and prebiotics (human milk oligosaccharides [HMOs] consumed only by bacteria, including specific HMOs, which encourage the growth of the beneficial Bifidobacterial species). These all serve to modulate the developing infant intestinal microbiota.¹⁰

Exclusively or partially breastfed infants, in contrast to exclusively formula-fed infants, appear more likely to be colonised by Lactobacillus and Bifidobacillus species and have lower levels of others such as C difficile, E coli, E cloacae and Citrobacter.^8,9

The microbiota of both breastfed and formula-fed babies changes with age and the introduction of solid foods, however a recent detailed metagenomics study indicates that it is the cessation of breastfeeding at any age, rather than the introduction of solid foods, that most dramatically alters the infant microbiome towards a more adult-like composition.^9,11

The effects of antibiotics

Antibiotic exposure in the first year of life is common in Western countries, including in Ireland, despite evidence that a significant number of prescriptions are unnecessary. In addition, the unnecessary use of broad spectrum antibiotics (where narrow spectrum would suffice) persists. What then do we know of the potential effects of antibiotic exposure on the developing infant intestine? 

Antibiotics, particularly broad spectrum antibiotics, result in the elimination of groups of bacteria and therefore a reduction in biodiversity of the microbiome. These gaps will be filled, possibly by other commensals, but also potentially by a growth of previously out-competed pathogenic organisms, rendering the host more susceptible to infection during the recovery period. 

A recent review by Vangay et al suggests that the effects of a single course of antibiotics may be fully ‘repaired’ with time and result in ultimate development of a normal adult profile. However, the possibility remains that in some cases long term imbalances in the microbiome may persist into adulthood.¹⁰ It is difficult to predict the effect in each individual given the multitude of other variables affecting microbiome development at this age.

In addition, to altering the ecology of the microbiome, antibiotics apply a positive evolutionary pressure to antibiotic resistance genes. These genes have been found to be present in intestinal bacteria of antibiotic naïve infants and children, presumably following vertical inheritance from the mother. 

Horizontal transmission of resistance genes between different species of bacteria has also been shown to occur, leading to a long term reservoir of multi-drug antibiotic resistance in the human gut.¹²

Indeed, gene based, culture independent techniques have demonstrated that the prevalence and number of antibiotic resistance genes in the intestinal microbiome has been significantly underestimated until now.¹³

What does the microbiota do?

Metagenomics has allowed us to understand more fully some of the roles of the intestinal microbiota. We know that commensal bacteria aid in the digestion of nutrients, including complex carbohydrates and indigestible fibres, which are then absorbed by the human host. Commensal bacteria also express a variety of enzymes that synthesise vitamins (eg. folate, B vitamins) and hormones (eg. melatonin) with effects on the human host.¹⁴

In addition, the interaction between gut microbes and the developing immune cells located in the lamina propria of the gut is necessary for the maturation of the innate and specific immune system, specifically the maturation and differentiation of T and B cells (including the Th2 cell profile and IgE regulation, both critical to allergic and atopic responses), the development of tolerance to commensal bacteria, the development of antigen specific defences against pathogenic bacteria, and the maintenance of an intact gut epithelial barrier. The first six months of life appear to be most critical for the development of a complete and normal immune system.¹⁰

The relationship between infant microbiota and disease: does it matter?

Given these complex and important roles for the intestinal microbiota, it is not surprising that much current research is focusing on the potential role of the microbiota in disease states. 

Obesity is a major public and personal health concern with a sharply rising incidence in Ireland and worldwide. Although not affecting infants, the age at which children are at risk of obesity is constantly decreasing.¹⁵ Animal studies support the role of antibiotic-triggered microbiome dysfunction in the development of obesity. Mice treated with antibiotics before or soon after weaning have a microbiota with a higher capacity to harvest calories from undigested luminal contents, as demonstrated by lower caloric output (ie. wastage in faeces despite similar caloric intakes), and higher adiposity than controls. 

Faecal transplantation from the antibiotic treated mice into germ free mice made the latter obese, further supporting the hypothesis of a dysfunctional microbiome contributing to obesity in mice.¹⁶ There is some confirmatory epidemiological evidence from human studies that individuals receiving broad spectrum antibiotics before the age of six months are at higher risk of obesity in later childhood, but multiple confounders make these data harder to interpret.^17,18,19

As outlined above, antibiotics are not the only modifier of the gut microbiota, and studies in both mice and human subjects seem to show that the ‘Western diet’ high in simple sugars and fats can modify the microbiota to one more efficient in the extraction of nutrients, thereby increasing the risk of obesity from two angles. Reverse modification of the diet by restricting fats and carbohydrates can however result in beneficial modifications to the metabolic profile of the microbiome, with positive effects on adiposity in studies of adult humans.^20,21

Allergic and atopic diseases

Allergic and atopic diseases also represent a significant public health burden with increasing prevalence in western societies. Although host genetics certainly play a role, the rapid increase over more recent years points to additional environmental triggers. A multitude of studies indicate that aberrations of the infantile gut microbiome (as well as airway microbiome), related to caesarean delivery, formula feeding and early antibiotic exposure, may be responsible for this increase. Specifically, higher levels of C difficile, E coli and lower levels of Bifidobacteria are linked to higher rates of IgE-associated eczema, and separately, caesarean birth and infantile antibiotic exposure are linked to higher rates of atopic diseases in early childhood.¹⁴ Studies in mice have shown that a germ free intestinal environment results in elevated serum IgE levels and an increased susceptibility to oral triggering of anaphylaxis than in mice with a diverse intestinal microbiota. Additionally, there is an inverse correlation between intestinal bacterial diversity levels and IgE levels in early life; this correlation is absent in adult mice, lending support to the time and age critical nature of the gut microbiome’s role in the development and maturation of the immune system.²²

Necrotising enterocolitis

Necrotising enterocolitis (NEC), a major cause of morbidity and mortality in premature infants, has been linked to intestinal dysbiosis. The acquisition of a ‘normal’ infant microbiome is impaired in premature infants, due to delayed feeding, high rates of antibiotic use and diminished acquisition of maternal intestinal bacteria. We know that formula feeding and prolonged antibiotic use are both independent risk factors for NEC in premature infants, and this may be due to their effect on the developing microbiota. Studies have shown differences between the microbiota of term and preterm infants and, significantly, differences between the microbiota of preterm infants who do or do not go on to develop NEC.²³

Microbiome alterations also have been implicated in the development of other conditions. For example, the development of IBD (Crohn’s disease and ulcerative colitis) is critically dependant on the intestinal microflora and many studies point to alterations in the microbiome of individuals with inflammatory bowel disease (IBD) that may be causal. The causative role of infection and inflammation in sudden infant death syndrome (SIDS), and the protective or detrimental immunomodulatory effect of the microbiome, also has been proposed.²⁴ Other conditions in which an aberrant microbiota has been causatively implicated include coeliac disease, type 1 diabetes and non-alcoholic fatty liver disease, among others.

Therapeutic and preventative strategies

It would appear then that targeting the intestinal microbiome not only has a sound hypothetical basis but also represents an attractive therapeutic and preventative strategy. Approaches being investigated include probiotics, prebiotics or even faecal transplantation. However, apart from a few isolated conditions, there is as yet no strong evidence from human studies that manipulating and ‘correcting’ the infant microbiome might have a therapeutic or prophylactic role in any or all of these diseases. Given the complexity of the development of the microbiome from birth to its ‘adult’ state around two to three years of age, it is difficult to predict what, if any, effect a disturbance to the microbiome will have on an individual’s risk of disease, and therefore difficult to select out the cohort that may benefit from a prophylactic intervention.

The term ‘probiotics’ refers to live commensal bacteria of different strains that are administered orally with the aim of inducing colonisation of the intestinal tract with beneficial effects. Examples of organisms typically used include Lactobacillus johnsonii and a variety of Bifidobacteria species. Although there is some preliminary evidence that early application of probiotics (before six months of age) may have a preventative effect on the later development of atopic conditions, it is not yet robust enough to warrant widespread clinical application. There has been much interest in probiotics for prophylaxis of NEC in premature infants. A recent meta-analysis demonstrated a reduction in incidence of severe NEC and mortality in premature infants treated with prophylactic probiotics.²⁵ As a result many neonatal units are now routinely administering probiotics to at risk infants, although there remains controversy about which probiotic species and dosage should be used.

Evidence is lacking to support the use of probiotics in the treatment or prevention of other conditions such as IBD, obesity and type 1 diabetes. Meta-analyses are limited by the low incidence of the target conditions and the heterogeneity of study designs (age of intervention, probiotic species and dose, and target population).²⁶

Prebiotics

Prebiotics are fermentable oligosaccharides, indigestible to humans but a nutrient source to encourage the proliferation of already existing specific bacterial species in the human intestine. Dietary fibres can also be broken down by intestinal bacteria into short chain fatty acids, which have a role both as an absorbable calorie source for the human host, as well as an immunomodulatory role through the promotion of gut epithelial integrity and the development of innate and specific host immunity.²⁷

A recent Cochrane review showed a decreased incidence of eczema in high risk infants treated with prebiotics.²⁸ There is a lack of evidence to confirm or refute the role of prebiotics in the prevention or treatment of other atopic or inflammatory conditions in the paediatric age group.

Faecal microbial transplantation

Faecal microbial transplantation (FMT) is another method for modifying the intestinal microbiome. Its first application was in the treatment of C difficile infection in adult patients, where it is of undoubted benefit.²⁹ The presumed mechanism of action is the modification of the recipient microbiome to align itself to the bacterial profile of the healthy donor; the addition of beneficial commensals may then outcompete the overgrown, pathological C difficile population. There is as yet limited evidence of benefit of FMT in the paediatric population and, as in studies of probiotics, there is a lack of conformity in study design (donor selection, dose, route of administration – nasogastric/colonoscopic, fresh/frozen transplant). Some trials suggest a therapeutic benefit in patients with IBD but all involve small numbers³⁰ and require further confirmation. There is no evidence of therapeutic benefit in other conditions and FMT is not yet ready to be routinely recommended in IBD or any other diseases in which an aberrant microbiome is implicated (apart from C difficile infection).

Our understanding of the intestinal microbiome

In summary, our understanding of the intestinal microbiome has expanded enormously over recent years. New techniques have permitted the identification of previously undetected bacteria and we know now that the microbiome is made up of tens of thousands of different species and comprises a total gene count 150-fold that of the entire human genome. It can be conceptualised as a discrete organ, with a multitude of roles both within and outside the gut, imbalance of which can be implicated in a broad range of pathologies. The period of infancy is especially critical in the development of a normal microbiome and this development may be disrupted by many different environmental factors with potentially long term implications for health. 

However, there are still many gaps in our knowledge, including defining ‘normal’ microbiome development and understanding the effect of alterations on different physiological and pathological pathways at different ages and stages of growth. While there are several observational human studies that support findings from interventional animal models of the relationship between dysbiosis and disease, large scale robust clinical trials are lacking. It will be interesting to observe developments and clarify which interventions, at what age and in which populations have a beneficial effect in both disease prophylaxis and therapy.

In the meantime an improved awareness of the complexity of the interaction between the intestinal microbiome and health should encourage us as clinicians to promote already proven health interventions in infants and young children, including breastfeeding, high fibre, low fat and low sugar diets, and the avoidance of unnecessary antibiotic use.

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